482
Biochemistry
TING-KAI LI, DAVID D. W R , AND BERT L. VALLEE
Anomalous Rotatory Dispersion of Enzyme Complexes. IV. Mechanism of Inhibition of Liver Alcohol Dehydrogenase by Buffer Anions and Bases* TING-KAILI, t DAVIDD. ULMER, $ AND BERTL. VALLEE From the Biophysics Research Labomtory of the Department of Medicine, Harvard Medical school, and the Peter Bent Brigham Hospital, Boston, Massachusetts Received December 6, 1962
The interaction of various buffer anions and bases with complexes which DPNH and 1,lOphenanthroline form with liver alcohol dehydrogenase has been studied by means of rotatory dispersion titration. At pH 7.5, pyrophosphate, chloride, formate, tris(hydroxymethy1)aminomethane, and imidazole reversibly displace equivalent amounts of both DPNH and 1,lO-phenanthroline. apparently through interaction with the zinc atoms of the enzyme. These agents do not alter the optical rotatory dispersion of the enzyme itself. Borate ions, in addition to interacting a t the zinc sites, also react with the ribosyl residues of DPNH. These effects upon the formation of the enzyme-coenzyme complex appear to underlie the inhibitory action of buffer ions upon the catalytic activity of liver alcohol dehydrogenase. The catalytic activity of horse liver alcohol dehydrogenase is inhibited by a variety of reagents, including buffer anions and bases (Theorell et al., 1955). Mechanisms by which these inhibitions may be brought about have been inferred from kinetic studies. Measurements of the physicochemical effects of such agents upon the interaction of the enzyme with its coenzyme and/or substrate a t equilibrium constitute an alternate approach to this problem. We have reported previously that highly specific Cotton effects result from the formation of binary complexes of liver alcohol dehydrogenase with chromophoric coenzymes, coenzyme analogs, and site-specific reagents (Ulmer et al., 1961). The magnitudes of these Cotton effects serve as direct measures of the degree of binding of such agents to the active centers of the enzyme a t equilibrium-a method which we have termed rotatory dispersion titration (Li et al., 1962aj. We have now employed this procedure to study the mechanism of inhibition of liver alcohol dehydrogenase by buffer anions and bases. The results indicate that such inhibitions by buffer ions may be brought about through the reversible displacement of DPNH from the zinc atoms of the enzyme. A preliminary account of these data has been given (Li etaZ., 196213). MATERIALS AND METHODS Crystalline alcohol dehydrogenase of horse liver was obtained from C. F. Boehringer and Soehne, Mannheim, West Germany. Prior to use, the enzyme was dialyzed for 5 days against 0.1 M sodium phosphate buffer, pH 7.5, a t 4" to remove low-molecular-weight impurities which absorb radiation a t 280 mp. The concentration of protein was determined by measurement of the optical density a t 280 mp, based upon an absorbancy index of 0.455 mg-* cm2 (Bonnichsen, 1950). Molar concentrations were based on a molecular weight of 83,300 (Ehrenberg and Dalziel, 1958). The catalytic activity of the enzyme for both the oxidation of ethanol and the reduction of acetaldehyde *This work was supported by the Howard Hughes Medical Institute and by grants-in-aid from the National Institutes of Health of the Department of Health, Education and Welfare, No. H-3117 (C4) and GM-K3-8260 ClB, and the National Science Foundation. t Fellow of the Helen Hay Whitney Foundation. Research Career Development Awardee of the National Institutes of Health, Department of Health, Education and Welfare.
was measured spectrophotometrically as described previously (Vallee et al., 1359). The concentration of reduced diphosphopyridine nucleotide (fl-DPNH, Sigma Chemical Company) was determined by measurement of the absorbancy at 340 mp (Kaplan, 1960). Solutions of DPNH in metal-free distilled water were prepared fresh every few days from the desiccated stock powder and stored a t p H 6 and 4". Under these conditions no significant formation of inhibitors due to decomposition of DPNH (Fawcett et al., 1961) was noted. 1,lO-Phenanthroline monohydrate (G. F. Smith and Company) was dissolved in 0.1 M phosphate a t p H 7.5. Sodium dihydrogen phosphate, sodium pyrophosphate, sodium chloride, sodium borate, and imidazole (Eastman) were all reagentgrade chemicals and were used without further purification. Tris (hydroxymethy1)aminomethane (Sigma Chemical Company), primary standard grade, was extracted with sodium dithizone in carbon tetrachloride a t p H 8 to remove contaminating metals prior to use (Thiers, 1957). D-Ribose was obtained from the Nutritional Biochemicals Company. Stock solutions of all reagents were adjusted to p H 7.5 with sodium hydroxide or hydrogen chloride. Dialyses were carried out in precleaned (Hughes and Klotz, 1956) cellulose casings (Visking Company). The purScation of water and glassware has been described (Vallee and Hoch, 1955). Optical rotation was measured by means of a model 200s-80Q photoelectric spectropolarimeter with oscillating polarizer prism (0. C. Rudolph and Sons); a high-intensity, high-pressure mercury lamp (A-H6, General Electric Company j was used as a light source. Excellent stability of the lamp could be maintained by circulating cooled, deionized water through the quartzjacketed lamp housing. All measurements were performed in wide-bore, 5-cm semi-micro polarimeter cells with quartz end-plates (0. C. Rudolph and Sons). The temperature of the solutions was maintained a t 10 f 1" by circulating cooled water through the polarimeter housing from an external bath. A stream of nitrogen gas directed onto the polarimeter cell endplates prevented fogging. Rotational angles were measured by the method of symmetrical angles (Rudolph, 1955). Rotatory dispersion titrations of alcohol dehydrogenase with DPNHl and 1,lO-phenanthroline
* B-DPNH itself is optically active. However, within the spectral range and at the concentrations employed in these titrations, the optical rotation of the coenzyme is barely detectable, and is not altered by the addition of the inhibitors.
Vol. 2,No. 3, May-June, 1963
MECHANISM OF INHIBITION OF LIVER ALCOHOL DEHYDROGENASE
in the presence and absence of inhibitors were performed as described previously (Li et d.,196%). The corresponding polarimeter blanks did not contain enzyme but were otherwise identical with the experimental samples. All measurements were performed with a constant symmetrical angle of 5". The slit width of the monochromator was restricted to less than 0.2 mm. The maximum optical density of the sample solutions within the wave length regions examined was less than 1.8 units per 5-cm pathlength. The optical rotatory dispersion of the liver alcohol dehydrogenase-DPNH complex was measured over a range of wave lengths varying from 300 to 450 mp, and that of the liver alcohol dehydrogenase-1,lo-phenanthroline complex from 295 to 340 mp. Specif~crotations were calculated on the basis of enzyme concentrations and are precise to 1" in the titrations of the enzyme with DPNH and to f 3" in the titrations of the enzyme with 1 , l O phenanthroline a t the lower wave lengths.
p
*
RESULTS The amplitude2 of the Cotton effect of the liver alcohol dehydrogenase-DPNH complex a t 327 mp is directly proportional to the concentration of the bound DPNH (Li et d.,1962a). Pyrophosphate does not affect the optical rotatory dispersion of the enzyme, but it markedly alters that of the enzyme-coenzyme complex. The magnitude of the Cotton effect, characteristic of the complex, is reduced in proportion to the concentration of pyrophosphate present. Thus, in the presence of 0.2 M pyrophosphate at pH 7.5, the reduction in amplitude of the Cotton effect from 67" to 28" is equivalent to a displacement of 58% of the DPNH bound to the enzyme a t saturation (Fig. 1). The displacement of DPNH by pyrophosphate is reversible. As the concentration of DPNH is increased to 6 X 10 -5 M, the amplitude of the Cotton effect concomitantly increases from 28" to 48", an increase of 30% in binding (Table I). Since DPNH binds a t or
483
/
290
/
340 WAVELENGTH I N m p
3 90
FIG. 1.-The effect of pyrophosphate ion concentration on the optical rotatory dispersion of the liver alcohol dehydrogenase.DPNH complex. Specific rotation a t 10O , -[a]:, is plotted against wave length. Liver alcohol dehydrogenase, 1.66 mg/ml in 0.1 M phosphate, p H 7.5, lo", served as the control (- - -). Identical concentrations of enzyme plus 4 X M DPNH (-) were exposed to 0.05 M pyrophosphate (B) and 0.2 M pyrophosphate (A). In each instance, identical concentrations of all the reagents in the absence of the enzyme served as the polarimeter blanks. The addition of pyrophosphate to the control did not alter its optical rotatory dispersion.
TABLE I
EFFECT OF PYEOPHOSPHATE AND VARYING CONCENTRATIONS OF D P N H ON THE AMPLITUDE OF THE C ~ EFFECT N OF "HE LIVERALCOHOLDEHYDROGENASE-DPNH COMPLEXa
Pyrophosphate (MI -
DPNH x 105)
(M
Amplitude of Cotton Effect (degrees)
_ _ _ _ _ ~ ~
0.2 0.2 0 0
-
4 6 4 6
28 48
WAVELENGTH IN m p
67 67
~~
Liver alcohol dehydrogenase: phosphate, pH 7.5, 10". a
1.66 mg/ml h 0.1
M
near the zinc atoms of alcohol dehydrogenase (Vallee et al., 1959; Mahler et al., 1962) and pyrophosphate is known to complex zinc, i t was thought that pyrophosphate and DPNH might compete for the metal site of the enzyme. This hypothesis was examined further. The chelating agent 1,10-phenanthrobne binds specifically to the zinc atoms of alcohol dehydrogenase, inducing a characteristic Cotton effect a t 297 mp (Vallee and Coombs, 1959; b e r et al., 1961). Upon addi tion of 0.2 M pyrophosphate, the amplitude of this Cotton effect is reduced from 80" to 36", demonstrating that pyrophosphate also displaces l,l0-phenanthroline * T h e terminology is in accord with thaL (1960).
of
Djciass!
FIG. %-The effect of pyrophosphate ions on the magnitude of the Cotton effect of the liver alcohol dehydrogenase. 1.10-phenanthroline complex. The specific rotation a t lo", - [a];,is plotted against wave length. Liver alcohol dehydrogenase, 0.5 mg/ml, in 0.1 M phosphate, pH 7.5, lo", served as the control (- - -). Identical con,entrations of enzyme plus 6 X 10 s M 1,lO-phenanthroline ' 0 ) were exposed to 0.2 M pyrophosphate (A). Addition of pyrophosphate to the control did not alter its optical rotatory dispersion.
from the zinc atoms of liver alcohol dehydrogenae (Fig. 2 ; . Chloride, formate, Tris, and imidazole do not alter the optical rotatory dispersion of the enzyme itself. but they reduce the magnitudes of the Cotton effects )f the enzyme DPNH and of the enzyme 1.10phenarithroline complexes (Tables I1 and 111 The decwases in the magnitudes of these f'otton effects are i , {VL ~ functions of the conc.wtr:itioii of these agents.
484
Biochemistry
TING-KAI LI, DAVID D. U W R , AND BIZRT L. VALLEE
TABLE I11 THEEFFECT OF BUFFER IONS ON TEE AMPLITUDE OF THE COTTONEFFECT OF m LIVERALCOHOL DEHYDROGENASE~,~~-PE~ENANTHROLINE COMPLEX^ Buffer Concentration Buffer Chloride Formate TI+ Imidazole Borate Phosphate /
FIG.3.-The effect of borate and &ribose on the optical rotatory dispersion of liver alcohol dehydrogenase and on the magnitude of the Cotton effect of the liver alcohol dehydrogenase.DPNH complex. The specific rotation at lo", - [a];, is plotted against wave length. Liver alcohol dehydrogenase, 1.66 mg/ml, in 0.1 Y phosphate, pH 7.5, loo, was the control (- - -). Identical concentrations of enzyme plus 4 x 10-6 Y DPNH (-) were exposed to 0.01 Y borate (A) and 0.01 Y borate plus 0.07 Y ribose (m). Ribose, 0.07 IS, did not alter the Cotton effect of the enzyme-coenzyme complex (---). Addition of both ribose and borate to the control did not alter its optical rotation.
The resultant changes in the degree of enzyme-coenzyme binding are proportional to the degree to which these agents affect enzyme-1 ,lo-phenanthroline binding. Again, in each instance, an increase in the concentration of DPNH concomitantly increases the magnitude of the Cotton effect of the enzyme-coenzyme complex, and the reversibility of the process is thereby demonstrated. Phosphate ions, employed as a control, neither inhibit enzymatic activity nor alter the TABLE I1 EFFECTOF BUFFERIONAND DPNH CONCENTRATIONS ON THE AMPLITUDE OF THE COTTON EFFECTOF THE LIVER ALCOHOL DEHYDROGENASE-DPNH COMPLEX^ ~
Buffer Concentration (M)
~-~ ~~
DPNH Concentration i~x 105)
Amplitude of Cotton Effect (degrees) ~~
Chloride
Formate
Tris
0 5 0 2 0 2 0 5 0 2 0.2 0.4 0.2
4 4 6 4 4
6 4
4 6
0.2
Imidazole
Borate
Phosphate
0 1 0 1 0 05
6
0 1 0 01
4 6
4 4
0 01
4
0 4
6
0 4
_____
0.5 0.5 0.4
0.1 0.1 0.005 0.4
44 33 40 29 44
80 80
a Liver alcohol dehydrogenase, 0.5 mg/ml, and 1,lOphenanthroline, 6 X 10-6 Y, in 0.1 Y phosphate, p H 7.5.
/
Buffer
(MI
Amplitude of Cotton Effect (degrees)
4
6
0 005
Liver alcohol dehydrogenase: phosphate, pH 7.5.
~
----
-
34 42 61 29 35 54 32 38 57 26 40 37 0 32 25 67 67 67
1.66 m g / d in 0.1 M
magnitudes of the Cotton effects of alcohol dehydrogenase complexes with DPNH and 1,lO-phenanthrohne (Tables I1 and 111). The effect of borate on these systems differs from that described for the ions above. The concentrations of borate which are required to displace DPNH from alcohol dehydrogenase are much lower than those needed to displace 1,lO-phenanthroline (Tables I1 and 111). The cause of this differential effectiveness was investigated further. Borate ions from complexes with sugars containing hydroxyl groups in the cis-co&guration (Boeseken, 1949). Though specific data demonstrating the interaction of ribose with borate could not be found in the Literature, such a reaction, demonstrated in the course of these studies (vide infra), might have been anticipated. Thus the interaction of borate with the ribosylhydroxyl groups of DPNH might well interfere with the formation of the enzyme-coenzyme complex. ~ e not affect Consistent with this hypothesis, ~ - r i b o does the binding of DPNH to alcohol dehydrogenase but it markedly curtails the effectiveness of borate ions in displacing bound DPNH (Fig. 3). The stoichiometry of the ribose-borate and DPNHborate complexes was determined by means of the difference in the optical rotation of the sugars and of their borate complexes (Zittle, 1951). A t p H 7.5, one mole of borate reads with each mole of ribose. Titration of DPNH with borate shows similar changes in optical rotation, which become maximal a t 2 moles of borate per mole of DPNH (Fig. 4). Apparently one mole of borate reads with each of the two ribosyl residues of DPNH, in accord with the stoichiometry of the ribose-borate complexes. DISCUSSION Inhibition of horse liver alcohol dehydrogenase by buffer anions and bases has been studied extensively. The kinetics of the inhibitions resulting from various buffer ions differ quantitatively and in certain instances even qualitatively (Theorell et al., 1955; Theorell and McKinley McKee, 1961; Plane and Theorell, 1961). Common mechanistic denominators to reconcile these apparent discrepancies have &?en sought (Theorell and McKinley McKee, 1961). The effects of these inhibiting ions upon the interaction of the coenzyme and other site-specific reagents with the enzyme are here examined a t equilibrium, an approach similar to that utilized to advantage previously in the study of the Old Yellow Enzyme (Theorell and Nygaard, 1954; Nygaard and Theorell, 1955). DPNH binds a t or near, and the chelating agent 1,lO-phenanthrohe binds directly to, the zinc atoms
Vol. 2, No. 3, May-June, 1963
of alcohol dehydrogenase (Vallee et d.,1959; Vallee and Coombs, 1959). Binding of these chromophoric molecules a t the active centers of the enzyme results in characteristic Cotton effects, the magnitudes of which are directly proportional to the concentrations of the complexes formed (Ulmer et d . ,1961; Li et d . ,1962a). Hence, these specific Cotton effects may be employed to measure the effect of a variety of inhibitors upon the binding of DPNH and of 1,lO-phenanthrohe to alcohol dehydrogenase, and to discern the interaction of these inhibitors a t the zinccontaining active centers of the enzyme. Pyrophosphate, chloride, formate, T r i s , 3 and imidazole, ions which inhibit the enzyme, all reversibly decrease the binding of DPNH and of 1,lO-phenanthroline (Figs. 1 and 2, Tables I, 11, 111). A given concentration of each inhibiting ion displaces DPNH and 1,lOphenanthrohe to the same degree; none of them alters the optical rotatory dispersion of the protein itself. It seems improbable, therefore, that the dissociation of the enzymecoenzyme and enzymechelate complexes is brought about through alterations in protein structure (Schellman and Schellman, 1958; Blout, 1960). Phosphate ions, which do not inhibit alcohol dehydrogenase, do not alter the characteristics of the Cotton effects. Hence, nonspecific effects, e.g. ionic strength, also do not Seem to underlie these observations. It would appear that the reversible displacement of both DPNH and 1,lO-phenanthroline from the enzyme is the consequence of the interaction of the anions and bases with the zinc atoms of the enzyme. Apparently all of these ions compete with the coenzyme for binding a t the active centers of alcohol dehydrogenase. The displacement of DPNH by these buffer ions is reminiscent of the effects of alkaline pH on the binding of DPNH. At p H 10, only one mole of DPNH is bound to each mole of the enzyme. The ionization of a specific group on the protein, perhaps a sulfhydryl group, which might be responsible for binding of the coenzyme was considered to be a possible explanation for this decrease in binding (Theorell and Bonnichsen, 1951). Detailed examination of the binding of DPNH to liver alcohol dehydrogenase as a function of p H has recently been performed by means of optical rotatory dispersion titration and fluorescence polarimetry (Li et al., 1962a; Brand et d., 1962). Two moles of DPNH remain bound to each mole of enzyme between pH 7.0 and 9.5, the amount decreasing sharply thereafter until none is bound a t p H 11. The present data suggest that the dissociation of the liver alcohol dehydrogenase-DPNH complex a t these p H values might be attributable to the interaction of hydroxyl ions with the zinc atoms of the enzyme in a manner similar to that shown here for buffer anions and bases. In fact, in the presence of increasing concentrations of Tris the enzyme-coenzyme dissociation curve measured as a function of p H shifts to progressively lower p H values, as might be expected from the combined effeds of both hydroxyl and Tris ions when interacting with the zinc atoms of the enzyme (Fig. 5). Compared to the other anions and bases studied, borate ions are disproportionately more effective in displacing DPNH than 1,lO-phenanthroline from the enzyme. The dual action of this agent can be held accountable for the disparity. Borate appears to interfere with the interaction of the enzyme with the coenzyme both by competing for the zinc atoms of the enzyme (Table 111) and by interacting with the ribose moiety of the coenzyme itself (Fig. 4). The latter 3
Unpublished data.
485
MECHANISM OF INHIBITION O F LIVER ALCOHOL DEHYDROGENASE
I60
___.
1
,
--
I 2 3 4 MOLES BORATE/MOLES DPNH
0
--_
FIG. 4.-ROtatOry dispersion titration of DPNH with borate ions. 5 X lo-' M DPNH in water at loo was titrated with increments of 0.2 Y sodium borate. The specific levorotation of the reaction mixture at 406 mp and loo, -[a]&, is plotted against the moles of borate added per mole of DPNH.
60 I
k
2
5
'1 A-in0.1MP04 11
.--In
"
1 +-in
oil
~
"
1
+ O 0 5 M TRlS 0 45M TRlS
+
\A
'4 \A
1
8.0
1
m\
9.0 PH
10.0
1I I i.0
FIG.&-The effect of alkaline pH and varying concentrations of tris(hydroxymethy1)aminomethane on the binding of DPNH to liver alcohol dehydrogenase. The amplitude of the Cotton effect of the enzyme.DPNH complex (see Fig. 1) is plotted against pH. Liver alcohol dehydrogenase, 1.66 mg/ml, and 6 X lod6Y DPNH in 0.1 Y PO4, 10' (A), were exposed to 0.05 116 "ria (m) and 0.45 36 "ria (0) at the pH values indicated.
reaction has also been suggested previously as a possible mechanism for the inhibition of borate ions in the yeast alcohol dehydrogenase system (Roush and Gowdy, 1961). The striking effectiveness of imidazole in displacing DPNH from liver alcohol dehydrogenase under equilibrium conditions bean3 upon previous studies. Imidazole has been shown to compete with DPN, ethanol, and acetaldehyde. Even though imidazole forms complexes with zinc (Koltun et d., 1958))it does not compete with DPNH under conditions appropriate for kinetic measurements (Theorelland McKinley McKee, 1961). Under conditions of equilibrium, the competition of imidazole with DPNH for binding to the enzyme becomes apparent, as shown by the present data (Tables I1 and 111).
486
D. ROBINSON, D. SPOLLAR, S. WHITE, A N D N. 0. KAPLAN
Rotatory dispersion titration provides a convenient method for the study of site-specific interactions of chromophoric coenzymes, coenzyme analogs, and inhibitors with appropriate enzymes (Ulmer and Vallee, 1961; Ulmer et al., 1961). The effect of optically inactive competitive agents on these optically active chromophoric complexes greatly extends the usefulness of this procedure, which can thus be employed to advantage for the study of enzymatic mechanisms.
REFERENCES Blout, E. R. (1960), in Optical Rotatory Dispersion, Djerassi, C., editor, New York, McGraw-Hill Book Company, p. 238. Boeseken, J. (1949), Aduan. Carbohydmte Chem. 4, 189. Bonnichsen, R. K. (1950), Acta Chem. S c a d . 4, 715. Brand, L., Everse, J., and Kaplan, N. 0. (1962), Biochemistry 1, 423. Djerassi, C., editor (1960), Optical Rotatory Dispersion, New York, McGraw-Hill Book Company. Ehrenberg, A., and Dalziel, K. (1958), Acta Chem. Scand. 12, 465. Fawcett, C. P., Ciotti, M. M., and Kaplan, N. 0. (1961), Biochim. Biophys. Acta 54, 210. Hughes, T. R., and Klotz, I. M. (1956), in Methods of Biochemical Analysis, vol. 3, Glick, D., editor, New York, Interscience Publishers, Inc., p. 265. Kaplan, N. 0. (1960), in The Enzymes, ed. 2, vol. 3, Boyer, P. D., Lardy, H., and Myrback, K., editors, New York, Academic Press, Inc., p. 105. Koltun, W. L., Dexter, R. N., Clark, R. E., and Gurd, F. R. N. (19581, J. Am. Chem. SOC. 80, 4188. Mahler, H. R., Baker, R. H., and Shiner, V. J. (1962), Biochemistry 1,47.
Biochemistry
Li, T. K., Ulmer, D. D., and Vallee, B. L. (1962a), Biochemistry 1, 114. Li, T. K., Ulmer, D. D., and Vallee, B. L. (1962b), Abstracts of Papers, 141st Meeting of the American Chemical Society, p. 55C. Nygaard, A. P., and Theorell, H. (1955), Acta Chem. Scand. 9, 1587. Plane, R. A., and Theorell, H. (1961), Acta Chem. Scand. 15, 1866. Roush, A. H., and Gowdy, B. B. (1961), Biochim. Bwphys. Ac& 52, 200. Rudohh. H. (1955). J. ODt. Soc. A m . 45. 50. Schell&&, C.‘G., A d Schellman, J. A. (1958), Compt. rend. trav. lab. Carkberg. Ser. Chim. 30, 463. Theorell, H., and Bonnichsen, R. (1951), A d a Chem. Scand. 5, 1105. Theorell, H., and McKinley McKee, J. S. (1961), Acta Chem. Scand. 15, 1834. Theorell, H., and Nygaard, A. P. (1954), Acta Chem. Stand. 8, 1649. Theorell, H., Nygaard, A. P., and Bonnichsen, R. (1955), Acta Chem. Scand. 9, 1148. Thiers, R. (1957), in Methods of Biochemical Analysis, v01. 5, Glick, D., editor, New York, Interscience Publishers, p. 273. m e r , D. D., Li, T. K., and Vallee, B. L. (1961), Pm. Nat. Acad. Sei. U . S. 47, 1155. m e r , D. D., and Vallee, B. L. (1961), J . BwZ. Chem. 236, 730. Vallee, B. L., and Coombs, T. L. (1959), J. Bwl. Chem. 234, 2615. Vallee, B. L., and Hoch, F. L. (1955), Pm. Nat. Acad. Sei. U. S. 41, 327. Vallee, B. L., Williams, R. J. P., and Hoch, F. L. (1959), J. Biol. Chem. 234, 2621. Zittle, C. A. (1951), Advan. Enzymol. 12,493.
Structural and Catalytic Alterations of Dehydrogenases After Photooxidation* DWIGHT ROBINSON, DAVID STOLLAR, SHIRLEY WHITE,AND NATHAN 0. KAPLAN Fmm the Graduate Department of Biochemistry, Brandeis University, W a l h m , Masswhusettst Received September 26,1962
The properties of three dehydrogenases have been studied after photooxidation with methylene blue. The resulting changes in catalytic activity include changes in coenzyme speciscity and substrate inhibition characteristics, accompanied by changes in coenzyme binding and in some physical properties. Although significant changes in catalytic activity were seen after destruction of less than 2 to 3 moles of histidine per mole of enzyme, there also were evidences a t this point of changes in enzyme structure. The structural changes produced after photooxidation in these and, perhaps, in other enzymes limit the usefulness of this technique for studying the role of histidine a t the active sites of enzymes. However, some changes in catalytic properties can be correlated with changes in enzyme structure. Methods have been sought for the specific destruction of amino acids in proteins in order to determine the functional role of certain amino acids in enzymes. Photooxidation of proteins with methylene blue destroys several amino acids, but generally destruction of histidine and tryptophan begins first and is followed by destruction of tyrosine, cysteine, and methionine as photooxidation is continued (Weil and Buchert, 1951; Weiletal., 1951,1952,1953).
* This work was supported in part by pan& from the National Institutes of Health (CY-3611 and 2G-2121, the National ScienceFoundation (G20,C29),and from the American Cancer Society (P-77E, the Thomas S. Miller Memorial Grant for Cancer Research). t Publication No. 211 of the Graduate Department of Biochemistry, Brandeis University, Waltham, Mass.
Previous studies with photooxidation have implicated histidine in the active site of certain enzymes (Weil et al., 1952,1953; Millar and Schwert, 1962), since loss of enzyme activity occurred as histidine was being destroyed (Weil et al., 1952, 1953). However, it has been pointed out that changes in protein structure which can accompany photooxidation may also account for the changes in enzyme activity (Brake and Wold, 1960). We have subjected three dehydrogenases, horse liver alcohol dehydrogenase ( m H ) , Ibeef he& m-le lactic dehydrogenase (beef H LDH), and chicken he& LDH)t to lactic dehydrogenase (chicken photooxidation with methylene blue and have o k ~ e d changes in catalytic activity as measured by the ability of the enzymes to react with NAD and analogs of NAD
